The fungus Candida albicans has emerged as an important opportunistic nosocomial pathogen. Candida species are now the fourth most common cause of hospital-acquired bloodstream infections and are a major cause of morbidity and mortality in the veteran population. Despite improvements in antifungal therapy, the high attributable mortality rate due to Candida infections is no better than two decades ago, and diagnostically, the traditional blood culture has only a 50% sensitivity to detect invasive candidiasis. Thus, our ability to prevent, diagnose, and treat Candida infections is still in need of great improvement. In the effort to identify novel diagnostic and drug targets, a number of C. albicans virulence-associated proteins have been investigated, and many of these are secretory proteins such as the secreted aspartyl proteases (Saps) and phospholipases. In addition, considerable recent effort has been made to study Candida biofilms, another key component of Candida pathogenesis. These biofilms enable Candida to colonize a protected site from which dissemination of infection and protection from anti-fungal therapy can occur. Thus, our overall objectives are to: (i) determine which genes are critical for the trafficking and secretion of Saps and other virulence proteins, and (ii) define the role of secretion in biofilm formation. If we can identify these key genes, this information may prove invaluable for understanding the mechanisms of biofilm formation and for identifying novel drug targets. In previous studies, we examined the role of the pre-vacuolar secretory pathway in the secretion of virulence-associated proteins and biofilm formation in C. albicans, regulated by the vacuolar protein sorting genes VPS1, VPS4, and PEP12. Of great interest, a C. albicans pep12 mutant was defective in virulence in vivo, and formed a biofilm that dramatically fragmented with minimal disturbance. Expanding on these studies, we will examine the late stages of secretion by examining key final steps in exocytosis regulated by the Exocyst protein complex, and determine its contribution to biofilm formation and virulence. Next, we will examine the molecular mechanisms of biofilm formation by investigating the critical determinants of biofilm integrity in the pep12 mutant. Therefore, this project will examine the key hypotheses that: (i) C. albicans exocytosis mutants missort proteins to the extracellular space resulting in defective biofilm formation, and (ii) mutations in secretory pathway genes at key steps in exocytosis will result in attenuated virulence in vivo, and (iii) C. albicans PEP12 is a key mediator of biofilm integrity. Specific Aim 1 is to determine the effects of mutations in C. albicans exocytosis genes at key steps of polarized secretion and their contribution to biofilm formation. Specific Aim 2 is to directly examine the requirement of C. albicans exocytosis genes for virulence in vitro and in vivo. Specific Aim 3 is to determine whether downstream effectors of C. albicans PEP12 are required for maintenance of biofilm integrity. Thus, our overall objectives are to define the key trafficking genes and pathways of these important secreted virulence proteins, and define their role in biofilm formation in order to understand mechanisms of pathogenesis and identify novel drug targets.